Antihypertension Nanoblockers Increase Intratumoral Perfusion of Sequential Cytotoxic Nanoparticles to Enhance Chemotherapy Efficacy against Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC), one of the worst prognosis types of tumors, is characterized by dense extracellular matrix, which compresses tumor vessels and forms a physical barrier to inhibit therapeutic drug penetration and efficacy. Herein, losartan, an antihypertension agent, is applied as a tumor stroma modulator and developed into a nanosystem. A series of lipophilic losartan prodrugs are constructed by esterification of the hydroxyl group on losartan to fatty acids. Based on the self‐assembly ability and hydrodynamic diameter, the losartan‐linoleic acid conjugate is selected for further investigation. To improve the stability in vivo, nanoassemblies are refined with PEGylation to form losartan nanoblocker (Los NB), and administered via intravenous injection for experiments. On murine models of pancreatic cancer, Los NB shows a greater ability to remodel the tumor microenvironment than free losartan, including stromal depletion, vessel perfusion increase, and hypoxia relief. Furthermore, Los NB pretreatment remarkably enhances the accumulation and penetration of 7‐ethyl‐10‐hydroxycamptothecin (SN38)‐loaded nanodrugs (SN38 NPs) in tumor tissues. Expectedly, overall therapeutic efficacy of SN38 NPs is significantly enhanced after Los NB pretreatment. Since losartan is one of the most commonly used antihypertension agents, this study may provide a potential for clinical transformation in stroma‐rich PDAC treatment.


Synthesis of losartan prodrugs
Scheme S1. Synthetic scheme of 1.

Determination of EE and DL
The encapsulation efficiency (EE) and drug loading (DL) contents of losartan in nanoparticles were determined by high-performance liquid chromatography (HPLC). The nanoparticle solutions were centrifuged at 4000 rpm for 5 min. Then, the supernatants were collected for Los-LA determination by HPLC. HPLC was performed using a Hitachi Chromaster 5000 system with a YMC-Pack ODS-A column (5 μm, 250 × 4.6 mm) at a flow rate of 1.0 mL/min. UV detection was performed at 220 nm. All of the runs used linear gradients of acetonitrile (solvent A) and water (solvent B) containing 0.1% trifluoroacetic acid (TFA). The EE and DL values were calculated according to Equations (1) and (2): (2) WLos in NPs, Wfed, and Wtotal represent the weights of losartan formulated into nanoparticles, the initial prodrugs fed for encapsulation, and the total amount of nanoparticles, respectively.

Characterization of nanoparticles by DLS
The hydrodynamic diameters of the nanoparticles were characterized through dynamic light scattering (DLS) measurements on a Malvern Nano-ZS90 instrument (Malvern, UK) at 25 ℃. Each sample was measured three times.

Morphological study of nanoparticles by TEM
The sample solution of Los-LA prodrug-loaded NPs at a concentration of 0.5 mg/mL (losartan equivalent) was dropped onto a 400-mesh copper grid coated with carbon. After 5 min of deposition, the surface liquid was removed with filter papers. Samples were stained with 2 wt% aqueous uranyl acetate solution for 1 min of positive staining and air-dried. The morphology of the nanoparticles was characterized using TECNAL 10 (Philips) at an acceleration voltage of 80 kV.

In vitro losartan release kinetics
Three milliliters of solutions containing free losartan@DSPE-PEG2000 micelles or Los-LA prodrug-loaded nanoparticles (0.1 mg/mL losartan equivalent concentration) in phosphatebuffered saline (PBS) (PBS and PBS containing 30 U/mL or 60 U/mL porcine liver esterase (PLE)) were loaded into dialysis bags (spectrum, molecular weight cutoff of 7 kDa) and dialyzed against 20 mL of releasing medium (PBS, pH 7.4, 0.4% Tween 80). The dialysis bags were continuously shaken in an orbital shaker (100 rpm) at 37 ℃. At predetermined time points, the releasing medium was collected to evaluate released losartan and Los-LA by HPLC.

Pharmacokinetic analysis of Los NB
Pharmacokinetic analysis was performed on Sprague-Dawley (SD) rats (male, 8 weeks old, n = 3/group). A single dose of free losartan or Los NB (20 mg/kg losartan equivalent dose) was intravenously injected via the tail vein. At predetermined time points, blood samples were taken following centrifuging at 3000 rpm for 10 min to gain plasma samples. The drugs were extracted by acetonitrile and the concentration was determined by liquid chromatography mass spectrometry (LCMS).

In vitro cytotoxicity analysis
Cells were planted in 96-well plates with the density of 4000-6000 cells per well. After 12 h, Drugs were added to the cells at various concentrations and incubated for an additional 24 h. Cell viability was investigated by CCK-8 assay. CCK-8 solution was added in each well (1:10, v/v) and continuously incubated for 1 h. The absorbance was measured at 450 nm using a microplate reader (Multiskan FC, Thermo Fisher Scientific). Cell viability (%) = absorbance of each well / absorbance of control well × 100%.

Cellular esterase activity analysis
Esterase activity of Panc02 and NIH-3T3 cells was measured with fluorescein diacetate (FDA), a cell-permeating esterase substrate. FDA was dissolved in DMSO at 10 mg/mL as a stock solution and diluted by PBS to 1 mg/mL as a working solution. Cells were planted in 96-well plates with the density of 1×10 4 cells per well. After 12 h, FDA working solution was added in each well (20 μg/mL final concentration) and the cells were incubated in dark. FITC fluorescence intensity was detected at frequent intervals using a microplate reader (Varioskan LUX, Thermo Fisher Scientific) (Ex = 480 nm, Em = 530 nm). For fluorescence imaging of esterase activity, cells were planted in confocal dishes with the density of 1×10 5 cells per dish. After 12 h, FDA working solution was added (5 μg/mL final concentration) and incubated for 30min in dark before observation.

Enzyme-linked immunosorbent assay (ELISA)
Panc02 and NIH-3T3 cells were planted in 12-well plates with the density of 1×10 5 cells per well.
After 12 h, culture medium was removed and replaced with serum-free medium (to decrease the cross-reactivity from FBS in ELISA), and cells were incubated with PBS or the same concentration of free losartan/Los-LA/Los NB (20 μM losartan equivalent) for 24 h. The supernatants were collected and the secretion level of TGF-β was determined through an ELISA kit (No. KE10005, Proteintech, China).

Immunofluorescence
Panc02 and NIH-3T3 cells were planted in confocal dishes with the density of 5×10 4 cells per dish.
After 12 h, cells were incubated with PBS or the same concentration of free losartan/Los-LA/Los NB (20 μM losartan equivalent) for 24 h. After being fixed in 4% PFA, Panc02 cells were stained with TGF-β antibody, and NIH-3T3 cells were stained with α-smooth muscle actin antibody (α-SMA) and fibronectin antibody. Cells were examined by optical microscope.

Multiplex tumor-infiltrating immunologic factors assay
Total protein of harvested Panc02 tumor tissue was extracted after ultrasonic grinding in PBS and centrifugation and was evaluated with a bicinchoninic acid (BCA) protein assay kit (Fdbio Science, Hangzhou, China). The tumor-infiltrating immunologic factors were evaluated using a LEGENDplex multiple assays kit (BioLegend, USA) according to the manufacturer's instructions.

Western blot analysis
Total protein of harvested tumor tissue was extracted after homogenization in RIPA buffer and centrifugation and was determined with a BCA protein assay kit. After separation on SDSpolyacrylamide gels, the proteins were transferred onto a PVDF membrane (Millipore, USA). The membrane was blocked with 5% bovine serum albumin for 1 hour and then incubated with primary antibodies at 4 ℃ overnight. The next day, the membrane was incubated with secondary antibodies for 1 hour at room temperature. Signals were detected using an ECL kit (Fdbio Science, Hangzhou, China). β-actin was used as a loading control.

Histological analysis
To evaluated the effect of Los NB on the tumor microenvironment, harvested tumor tissues were fixed in 4% PFA, embedded in paraffin, sectioned into 5 μm thick sections, and stained with αsmooth muscle actin antibody (α-SMA), fibronectin and hypoxia inducible factor-1α (HIF-1α) antibodies. Slides were examined by optical microscope.
To evaluate the effect of Los NB on tumor vessel perfusion, mice were intravenously injected with DyLight 488 Lycopersicon Esculentum (Tomato) Lectin (Vectorlab, USA) 5 minutes prior to sacrifice. Harvested tumor tissues were embedded and frozen in OCT, sectioned into 5 μm thick sections, and stained with CD31 antibody. Slides were examined by confocal microscopy (Olympus, FV3000, Japan). Tumor vessel perfusion was quantitatively evaluated by calculating the percentage of perfused vessels (CD31 colocalized with lectin) among total vessels (total CD31).
To detect the distribution of DiR-loaded SN38 NPs in tumors, tumor tissues were prepared into 5 μm frozen sections, stained with CD31 antibody, and examined by confocal microscopy.
To investigate the stroma, apoptosis and proliferation levels in tumors after antitumor treatment, sections were analyzed by hematoxylin and eosin (H&E), Masson's trichrome staining, TdTmediated dUTP nick end-labeling (TUNEL) and Ki-67 staining.
Five randomly chosen visual fields were quantitatively analyzed using ImageJ software.

RNA-sequencing Analysis
RNA sequencing was performed on the liver of mice undergoing different treatment. BALB/c nude mice were injected a dose of saline/free losartan/Los NB (20 mg/kg losartan equivalent) following a dose of saline/SN38 NP (8 mg/kg SN38 equivalent) after 24 h. At 24 h after SN38 NP injection, livers were harvested. Total RNA was extracted using Trizol reagent kit (Invitrogen, USA) according to the manufacturer's protocol. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, eukaryotic mRNA was enriched by Oligo (dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reversly transcribed into cDNA by using NEBNext Ultra RNA Library Prep Kit for Illumina          HIF-1α staining) in tumor tissues (n = 5). Five randomly chosen visual fields were evaluated for histological quantification. Data are presented as mean ± SD. Significance was assessed by oneway ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Figure S10. Immune microenvironment changes in Panc02 tumors after saline/free Los/Los NB treatments. A-F) Representative images and quantitative analysis of immunohistochemistry staining of CD3 + T cells, CD8 + T cells and F4/80 + macrophages (n = 5). G-I) Content of IL-2, IL-12p70 and TNF-α in Panc02 tumor tissues (n = 5). Five randomly chosen visual fields were evaluated for histological quantification. Data are presented as mean ± SD. Significance was assessed by one-way ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Figure S11. Representative TUNEL and Ki-67 staining images and quantitative analysis of Panc02 tumor tissues on Day 28 (n = 5). Five randomly chosen visual fields were evaluated for histological quantification. Data are presented as mean ± SD. Significance was assessed by one-way ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. quantification. Data are presented as mean ± SD. Significance was assessed by one-way ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Figure S13. Quantification of Masson and Sirius red staining of PDX tumor tissues on Day 28 (n = 5). Five randomly chosen visual fields were evaluated for histological quantification. Data are presented as mean ± SD. Significance was assessed by one-way ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001. Figure S14. Quantification of TUNEL and Ki-67 staining of PDX tumor tissues on Day 28 (n = 5). Five randomly chosen visual fields were evaluated for histological quantification. Data are presented as mean ± SD. Significance was assessed by one-way ANOVA followed by LSD post hoc test. ns, p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001.